The invention discloses an innovative method and apparatus by which the motion of charged particles in a solution subject to an applied electric field may be measured. Although the present invention will refer to macromolecules throughout much of its specification, the invention includes more generally all classes of small particles including emulsions, viruses, nanoparticles, liposomes, macro-ions and any other solution constituents whose size may lie between a half and a few thousand nanometers. Thus whenever the terms “molecule,” “macromolecule,” or “macro-ion” are used, it should be understood they include all of the aforementioned solution-borne objects.
The electrophoretic mobility is the directly measurable and most widely used quantity to characterize the charge of the molecules, or for that matter any other particles present, in such solutions. Once measured, the electrophoretic mobility can in turn be used to determine the effective charge, Ze, carried by such molecules as well as its so-called zeta potential ζ. The interface between the group of ions tightly bound to the particle and those of the surrounding solution that do not move with the particle defines the hydrodynamic shear plane. The zeta potential represents the electrostatic potential existing at this shear plane. It is a basic objective of the present invention to provide an improved means by which the electrophoretic mobility, effective charge, and zeta potential of molecules and particles in solution may be measured.
Electrophoresis is the migration of macro-ions under the influence of an electric field. A steady-state electrophoretic velocity, ve, attained by the migrating macro-ions is linearly proportional to the applied electric field. When a field is applied, the molecules' velocities are essentially always in equilibrium. One can measure the proportionality constant, in this case the electrophoretic mobility, by measuring the electrophoretic velocity and dividing by the applied electric field. An objective of the present invention is to provide an improved apparatus capable of measuring the electrophoretic velocity of particles in solution more accurately than has been possible heretofore.
Several techniques have been developed and are available for measuring mobilities. Among these techniques are the Moving Boundary Method, Microelectrophoresis, and light scattering methods such as heterodyne dynamic light scattering, DLS, laser Doppler electrophoresis, LDE, and phase analysis light scattering, PALS. The Moving Boundary Method employs a specialized cell to establish two sharp boundaries between the macromolecular solution and a buffer. The translation of the boundaries under the application of an electric field provides a quantitative means to determine mobilities. Microelectrophoresis is based on directly observing the electrophoretic motion of individual particles with magnifying optics. However, microelectrophoresis is limited to measurement of the mobilities of particles much larger than about 100 nm in radius. Light scattering techniques have also been developed for measuring electrophoresis. Light scattered from moving particles carries information relating to such motion and from which their electrophoretic mobility may be determined.
Consider now the most significant of these measurement techniques for the measurement of electrophoretic mobilities: PALS. A beam of monochromatic light, usually from a laser source, illuminates a sample of liquid borne particles exposed to an applied electric field. Some of the light they scatter is collected and combined with a fraction of the incident unscattered light. In other words, the scattered signal is combined coherently with the incident light to produce a heterodyned signal. Such combination of the two beams is generally achieved using a single mode fiber to insure good alignment between the two beams. This technique generates fringes with high contrast, but results in a combined beam of relatively low intensity as a large fraction of the energy of each component beam is lost to modes not supported by the fiber. It is another objective of the present invention to combine such beams in free space producing a combined coherent beam of far greater energy and a correspondingly simplified detector system.
In order to measure the fluctuations of the combined beams and derive therefrom measurement of the electrophoretic mobility of the scattering particles, one of the combining beams is directed to reflect first from an oscillating mirror. This causes the detected fringes to aquire an intensity modulation, even in the absence of electrophoretic motion. The electrophoretic motion that results from the application the applied field produces a frequency shift permitting, thereby, an unequivocal determination of the sign of the mobility. However, practical considerations, such as those discussed by Robert G. W. Brown in “Homodyne Optical Fiber Dynamic Light Scattering,” Appl. Opt. 40, 4004-4010 (2001), require the relative intensities of the two combined beams must be adjusted so that the ratio of the two is less than about 30. Another objective of the present invention is to remove the need to adjust the beams' relative intensities allowing such ratios to be greater than even 100:1 in order to take advantage of the effect known as coherent amplification, which will be described in greater detail in the section entitled Detailed Description of the Invention.
Because the combined beam intensity ratios of the traditional PALS measurement are restricted to a relatively small range, the detection of the signals produced thereby requires use of a photomultiplier, PMT, or an avalanche photodiode, APD. Such detectors are expensive and can be severely damaged or even destroyed by their inadvertent exposure to ambient room light or other extraneous sources. Another important objective of this invention is to eliminate these expensive components while at the same time providing detection means that cannot be damaged by extraneous light sources. Because PMT and APD devices have very limited dynamic ranges, the light intensity and the ratio of the two beams must be continually adjusted to keep the detector within their linear ranges. Alternatively one must correct of the effects of the nonlinearity. An objective of this invention is to incorporate a detection means with a wide linear response so that such adjustments and modeling are unnecessary.
It is another objective of this invention to measure the mobility of molecular species rapidly and without damage to the molecules being measured. A further and particularly important objective of this invention is to measure the mobility of molecules whose effective size is well below the PALS limit of about 5 nm. Still another objective of this invention is to increase the linear range of measurements possible to extend to lower concentration and size.